Method for isolating a target biological material, capture phase, detection phase and reagent containing them

ABSTRACT

The invention concerns a method for isolating a target biological material contained in a sample, which consists in providing a capture phase comprising an organic molecule having at least a reactive function and at least a protein material capable of recognizing or binding, specifically and directly or indirectly, with the target biological material, said protein material having a specific covalent binding site with the organic molecule reactive function, consisting of at least a tag comprising at least six contiguous lysine, or lysine derivative residues, the method consists in contacting said target biological material with at least the capture phase; and detecting the target biological material fixed on the capture phase: The invention also concerns the capture and detection phases, and a reagent containing them.

[0001] The invention relates to a method for isolating a targetbiological material contained in a sample, using a capture phasecomprising an organic molecule containing at least one reactivefunction, and at least one protein material capable of recognizing orbinding, specifically and directly or indirectly, to the targetbiological material, said protein material containing a specific sitefor covalently binding to the reactive function of the organic molecule.

[0002] A method for immobilizing a protein on a solid support, by meansof reactive functions, in order to stimulate the proliferation or growthof T cells and the production of killer lymphocytes is known fromdocument EP-A-0,319,012. To this end, the protein has, at its C-terminalend, a covalent-binding site for binding to said reactive functions,which consists of an amino acid sequence in which are distributed two tofour lysine residues.

[0003] The problem posed by the covalent-binding site of this prior artlies in the fact that, in a diagnostic application, the results of theisolation of a target biological material with which it is capable ofbinding are similar to those obtained with a protein material which doesnot contain such a binding site.

[0004] According to the invention, a method is provided for isolating atarget biological material by using at least one capture phase whichcomprises a protein material containing a covalent-binding site whichallows efficient orientation of said protein material, and leads tosensitive, high-quality detection of said biological material.

[0005] Thus, the method of the invention, for isolating a targetbiological material contained in a sample, comprises the followingsteps:

[0006] a capture phase is provided, which comprises an organic moleculecontaining at least one reactive function and at least one proteinmaterial capable of recognizing or binding, specifically and directly orindirectly, to the target biological material, said protein materialcontaining a specific site for covalent binding to the reactive functionof the organic molecule, which consists of at least one tag comprisingat least six contiguous lysine or lysine-based residues,

[0007] said target biological material is placed in contact with atleast one capture phase, and

[0008] the target biological material bound to the capture phase isdetected.

[0009] According to the invention, the expression “isolating abiological material” means the binding, separation, isolation, detectionand/or quantification of this material, the enrichment of a fractionwith target biological material, according to a qualitative and/orquantitative, specific or aspecific binding method.

[0010] According to one variant of the method, the capture phase canalso comprise a label, and, in this case in particular, it can consistof a detection phase.

[0011] According to another variant of the method, a detection phase isalso provided, which comprises an organic molecule containing at leastone reactive function, at least one protein material capable ofrecognizing or binding, specifically and directly or indirectly, to saidtarget biological material, and a label, said protein materialcontaining a specific site for covalent binding to the reactive functionof the organic molecule, which consists of at least one tag comprisingat least six contiguous lysine or lysine-based residues.

[0012] In this case, the organic molecules in the capture phase and inthe detection phase, respectively, can be identical or different, andthe protein materials in the capture phase and in the detection phase,respectively, can be identical or different.

[0013] As is understood by the invention, a sample comprises any samplecapable of containing a biological material, in particular a sample suchas that obtained from—a biological fluid, a sample of food origin, or acell culture.

[0014] The sample consists of all or part of another sample: inparticular it can consist of an aliquot or a dilution.

[0015] A protein material according to the invention comprises proteins,in particular recombinant proteins, especially antigens, antibodies andpeptides such as synthetic peptides. The method of the invention mayalso be carried out with a material such as peptide analogues of nucleicacids (PNA).

[0016] The term “organic molecule” means a molecule of variable size;thus, this refers equally to a small molecule such as a hapten and to amacromolecule such as a polymer.

[0017] As examples of haptens, mention may be made of a hormone, avitamin, such as biotin, or a medicinal product. In this case, themethod of the invention can comprise, before the step of detecting thetarget biological material, a step of binding the organic molecule to acarrier molecule. Preferably, the hapten is biotin and the carriermolecule is avidin.

[0018] A polymer as used according to the invention is a polymer inparticulate or in linear form. It can be a homopolymer chosen inparticular from polylysine and polytyrosine, or a copolymer chosen inparticular from maleic anhydride copolymers, N-vinylpyrrolidonecopolymers, natural or synthetic polysaccharides, polynucleotides andamino acid copolymers such as enzymes. Advantageously, it is a copolymerchosen from maleic anhydride/methyl vinyl ether copolymer,N-vinylpyrrolidone/N-acryloxysuccinimide copolymer, poly-6-aminoglucose,and enzymes such as horseradish peroxidase (HRP) or alkaline phosphataseor derivatives thereof bearing at least one reactive function.

[0019] The expression “reactive function of the organic molecule” meanseither a reactive function chosen in particular from ester,halocarbonyl, sulfhydryl, disulfide, epoxide, haloalkyl and aldehydefunctions; or a function which can be activated by an activating agentsuch as carbodiimides or homo- or heterobifunctional compounds. By wayof example, an activatable function is chosen in particular from acid,amine and hydroxyl functions.

[0020] The covalent-binding sites defined above can exist naturally inthe protein material. Alternatively, they can be “incorporated”beforehand into the protein material, in the form of a tag, according totechniques which are well known to those skilled in the art, such as thetechnique used to purify proteins by the IMAC (immobilized metalion-affinity chromatography) method on resins (1, 2). By way of example,such sites can be incorporated into a protein material, and inparticular a protein, by genetic engineering in order to obtainrecombinant proteins.

[0021] A tag can be defined as an amino acid sequence which isincorporated into, i.e. added to, the original structure of the proteinmaterial, which is introduced into a preferred place in said originalstructure in order to allow it to be exposed in a relevant manner, inparticular with regard to its covalent binding to the organic molecule.

[0022] In accordance with the invention, a covalent-binding site of theprotein material considered can consist of a tag comprising six or morelysine or lysine-based residues, and optionally other amino acids, orcan consist of several of said tags.

[0023] The term “lysine-based” means that the lysine can be chemicallymodified, provided that these modifications essentially preserve or evenenhance the specificity of the covalent-binding site. Examples which maybe mentioned are the replacement of L-lysine with D-lysine, andvice-versa; a modification of the lysine side chain, such as anacetylation of the amine function or an esterification of the carboxylfunction; a modification of the peptide bonds such as, for example,carba, retro, inverso, retro-inverso, reduced and methylenoxy bonds.

[0024] The tag(s) described above can be found in any place in theprimary structure of the protein material. Preferably, it is located atthe N- or C-terminal end of the protein material.

[0025] According to the present invention, various methodes are alsodefined for isolating a target biological material contained in asample, depending, in particular, on its nature:

[0026] if the target biological material is an antibody, the proteinmaterial comprises an antigen which specifically recognizes saidantigen;

[0027] if the target biological material is an antigen, the proteinmaterial comprises an antibody which specifically recognizes saidantigen.

[0028] Other subjects of the invention are outlined below.

[0029] Thus, the invention relates to a phase for capturing a targetbiological material which comprises an organic molecule containing atleast one reactive function and at least one protein material capable ofrecognizing or binding, specifically and directly or indirectly, to thetarget biological material, said protein material containing a specificsite for covalent binding to the reactive function of the organicmolecule, which consists of at least one tag comprising at least sixcontiguous lysine residues; preferably, the organic molecule is apolymer or a hapten.

[0030] An advantageous polymer is particulate or linear and chosen inparticular from homopolymers such as polylysine or polytyrosine; andcopolymers such as maleic anhydride copolymers, N-vinylpyrrolidonecopolymers, natural or synthetic polysaccharides, polynucleotides andamino acid copolymers such as enzymes. Even more advantageously, thepolymer is chosen from maleic anhydride/methyl vinyl ether copolymer,N-vinylpyrrolidone/N-acryloxysuccinimide copolymer, poly-6-aminoglucose,horseradish peroxidase (HRP) and alkaline phosphatase.

[0031] If the organic molecule is a hapten, the capture phase can alsocomprise a suitable carrier molecule. Preferably, the hapten is biotinand, if necessary, the carrier molecule is avidin.

[0032] Advantageously, the tag as defined above is placed at the N- orC-terminal end of the protein material.

[0033] The reactive function of the organic molecule is chosen inparticular from ester, acid, halocarbonyl, sulfhydryl, disulfide,epoxide, haloalkyl and aldehyde functions.

[0034] The invention also relates to a phase for detecting a targetbiological material, having the same characteristics as the abovecapture phase and also comprising a label.

[0035] As indicated above, the organic molecule can be a polymer or ahapten. According to the second possibility, the organic moleculeconsisting of the hapten also represents the label.

[0036] The label for the detection phase is preferably chosen from thegroup consisting of an enzyme, a protein, a peptide, an antibody, ahapten such as biotin or iminobiotin, a fluorescent compound such asrhodamine, a radioactive compound, a chemiluminescent compound, anelectron-density component, a magnetic component and the like.

[0037] Another subject of the invention is a reagent for isolating atarget biological material, comprising a capture phase of the inventionand/or a detection phase of the invention.

[0038] Advantageously, the capture phase is bound, directly orindirectly, to a solid support, by passive absorption or by covalency.

[0039] The solid support can be in any suitable form, such as a plate, acone, a bead which is optionally radioactive and/or fluorescent and/ormagnetic and/or conductive, a bar, a tube of glass, a well, a sheet, achip or the like. It is chosen from among polystyrenes,styrene/butadiene copolymers, styrene/butadiene copolymers mixed withpolystyrenes, polypropylenes, polycarbonates, polystyrene/acrylonitrilecopolymers, styrene/methyl methylmethacrylate copolymers, from amongsynthetic and natural fibres, and from among polysaccharides andcellulose derivatives, glass, silicon and derivatives thereof.

[0040] As will be demonstrated in the experimental section below, theAuthors have obtained monoclonal antibodies directed against the tags asdefined above and in particular against the tag which consists of asequence of six lysine residues.

[0041] It is thus possible to envisage the use of such a tag in a methodfor isolating a biological material, according to which:

[0042] a capture phase is provided, said target biological material isplaced in contact with at least the capture phase, and the targetbiological material bound to the capture phase is detected, according towhich method the capture phase comprises an organic molecule whichcomprises or consists of an anti-tag antibody and at least one proteinmaterial capable of recognizing or of binding, specifically and directlyor indirectly, to the target biological material, said protein materialcontaining a specific site for binding to the organic molecule, whichconsists of at least one tag comprising at least six contiguous lysineor lysine-based residues.

[0043] Such a method can also comprise the use of a detection phase,which has the characteristics of the capture phase as has just beendescribed, and also comprising a label. This label can advantageouslyconsist of a said antibody.

[0044] The characteristics and advantages of the various subjects of theinvention are illustrated below, in support of Examples 1 to 6 and FIGS.1 to 7.

[0045]FIG. 1 represents a graph reflecting the yield of coupling bycovalent binding between the protein RH24K (light-shaded bars) and theprotein RH24 (dark-shaded bars) and the copolymer, for differentcoupling conditions; Acet=acetate; Phos=phosphate; Bor=borate;Carb=carbonate.

[0046]FIG. 2 represents a graph of comparison between the stability as afunction of time (in days) of the polymer (MAVE)/protein (RH24K)conjugate compound (curve ▪) and that of the free RH24K protein (curve).

[0047]FIG. 3 represents a scheme summarizing the “sandwich” detectiontechnique.

[0048]FIG. 4 represents a graph reflecting the immunoreactivity of thepolymer/RH24 conjugate compounds (curve ), and polymer/RH24K conjugatecompounds (curve ▪), with respect to a monoclonal antibody.

[0049]FIG. 5 represents a graph reflecting the immunoreactivity of thepolymer/RH24 conjugate compounds (curve ) and polymer/RH24K conjugatecompounds (curve ▪), with respect to serum.

[0050]FIG. 6 illustrates the results of a sandwich and indirect ELISAtest with the antibody IG2D4, using a graph representing the OD as afunction of the antibody titer.

[0051]FIG. 7 illustrates the results of an indirect ELISA test with theantibody IG2D4, using a graph representing the OD as a function of theantibody titer.

EXAMPLE 1 Expression and Purification of RH24K

[0052] pMH24 (Cheynet et al., 1993) was modified by insertion of asynthetic adapter at the 3′ end of the p24 gene in order to expressRH24K, corresponding to RH24 which has at its C-terminal end a specificsite for covalent binding referred to as a “polylysine tail” andconsisting of 6 contiguous lysine residues.

[0053] The adapter was prepared beforehand by hybridization of twooligonucleotides having the following respective sequences:

[0054] SEQ ID NO: 1:5′ GGGAAGAAGA AGAAGAAGAA GTCTGTCGAC GAATCTCT 3′

[0055] SEQ ID NO: 2:5′ CTAGAGAGAT TCGTCGACAG ACTTCTrCTT CTTCTTC′UCC C3′in 10 mM Tris, 50 mM NaCl, 1 mM DTE, 10 mM MgCl₂, pH 7.5 buffer, byheating at 70° C. for 5 minutes followed by slow cooling first to 37° C.over 30 minutes and then to room temperature over 30 minutes. Thisadapter was then cloned between the sites SmaI and XbaI of pMH24 at the3′ end of the RH24 gene. The resultant plasmid, pMH24K3′, was amplifiedin the E. coli XLI bacterial strain (recA1, endA1, gyrA96, thi-1,hsdR17, supE44, relA1, lac [F′ proAB, lacIPZΔAM15, Tn10 (tet^(r))] andthen sequenced by the Taq Dye Deoxy Terminator Matrix Standart method(Applied Biosystems). The sequence obtained and the correspondingsequence on the protein obtained at the C-terminal are given below.          SmaI      5′ATG CCC GGG AAG AAG AAG AAG AAG AAG Nter Met . . .[247 amino acids] . . . Pro Gly Lys Lys Lys Lys Lys Lys                       XbaI TCT GTC GAC GAA TCT CTC TAG A 3′ Ser Val AspGlu Ser Leu       Cter

[0056] The protein is expressed under the control of the Tac promoter inthe E. coli XLI bacterial strain (see above). A preculture of E. coliXLI containing the expression vector, is prepared beforehand overnightat 37° C. in Luria broth (LB) in the presence of ampicillin (amp; 50μg/ml) and tetracycline (tet; 12 μg/ml) in order to inoculate theLB-amp-tet medium (1/40 to 1/25); this medium is cultured until an OD at600 nm of 0.6 is obtained. Expression of the protein is then induced byaddition of isopropyl-β-D -thiogalactopyranoside (IPTG) to the medium ata concentration of 1 mM for 2h30 at 37° C. with stirring. Aftercentrifugation at 5000 revolutions/min (JA21 rotor, Beckman) for 10minutes at 4° C., the bacteria are taken up in a 50 mM Tris-HCl, 1 mMEDTA, 100 mM NaCl, 10 mM MgC12, pH 8 buffer (3 ml/g of biomass) in thepresence of protease inhibitor (2 μg/ml aprotinine and 2 μg/mlleupeptine) and then lyzed by sonication at 0C. The soluble fraction istaken up by centrifugation at 10,000 revolutions/min (JA21 rotor,Beckman) for 15 minutes at 4° C. and then deposited on a metal-chelatingaffinity column in order to purify the RH24K (Chelating Sepharose FastFlow No. 17057501). The gel was preloaded with Zn²⁺ according to thesuppliers, recommendations and then equilibrated with a sodium phosphatebuffer containing 67 mM NaCl, pH 7.8, and then 200 mM ammonium acetate,0.5 M NaCl, pH 6. The RH24K is eluted with 200 mM ammonium acetate 0.5 MNaCl pH 4. The fractions collected are dialyzed against 50 mM, pH 7.8sodium phosphate buffer.

[0057] The purified protein is characterized by electrophoresis onSDS-PAGE and by passage in gel filtration/HPLC. Its purity is greaterthan 90% and thus comparable with the purity of RH24. When analyzed bymass spectrometry, its molecular weight is 28,027, which is compatiblewith the theoretical molecular weight 27,992. The protein is recognizedby polyclonal and monoclonal anti-p24 antibodies in Western Blot and inELISA.

EXAMPLE 2 Coupling of RH24K and of RH24 to the NAVE Copolymer

[0058] 5 μl of polymer solution at a concentration of 1 g/l is added toa solution of protein in a suitable buffer, at a concentration of 1 g/l.

[0059] The reaction medium is stirred for 3 hours at 37° C.

[0060] The reaction progress is monitored by HPLC, on a Waters stericexclusion column with a 0.1 M, pH 6.8 phosphate buffer as mobile phase.

[0061] The coupling yields reported in FIG. 1 show that the modifiedprotein couples preferably to the polymer and over a wider spectrum ofexperimental conditions.

EXAMPLE 3 Effect of the Nature of the Polymer on the Coupling Yield

[0062] The effect of the nature of the comonomer forming part of thecomposition of the polymer is evaluated under the same conditions asthose described above.

[0063] The results are given in the table below. TABLE Coupling yieldPolymer (%) MAVE 65 poly(methyl vinyl ether/maleic anhydride PEMA 86poly (ethylene/maleic anhydride) SMA 49 poly (styrene/maleic anhydride)NVPmA 36 poly (N-vinylpyrrolidone/ maleic anhydride

[0064] The effect of the comonomer is quite pronounced since thecoupling yields vary within a range between 36 and 86% depending on thenature of the comonomer.

EXAMPLE 4 Compared Stability of the Protein/Polymer Conjugate Compoundswith Respect to the Protein Alone

[0065] The conjugate compounds were stored at 37° C. in the couplingmedium and analyzed by steric exclusion chromatography in order toevaluate the amount of residual protein in the coupling medium. Theresults are given in the graph represented in FIG. 2.

[0066] The coupling on polymer thus gives the protein better stabilityon storage.

EXAMPLE 5 Directed Biotinylation of RH24K

[0067] The conjugate compounds prepared are biotinylated p24s (RH24 orRH24K) which will be used in detection in the p24 antigen sandwich test,in accordance with the scheme represented in FIG. 3.

[0068] The biotin couplings are carried out on RH24K and on RH24,checked and then analyzed in the antigen sandwich test in order todefine the coupling conditions which are favorable to RH24K.

[0069] The proteins are used at a concentration of 1 mg/ml. Differentbuffers were tested on the basis of the buffers tested during thep24/MAVE couplings: 0.1 M borate pH 9.2, 0.1 M Tris pH 7.2, 0.1 Mphosphate pH 7.2 and 0.1 M carbonate pH 8.5. The following biotin/p24ratios were tested: 5/1, 10/1, 25/1, 50/1, 100/1. The couplings wereincubated for 1 hour at 37° C.

[0070] The p24/biotin conjugate compounds were used in detectionaccording to the schema represented in FIG. 3 with monoclonal antibodiesand a positive serum.

[0071] The RH24/biotin and RH24K/biotin conjugate compounds showactivity in the antigen sandwich test with the monoclonal antibodies andthe positive serum. By way of illustration, FIGS. 4 and 5 show, in graphform, the compared responses of the conjugate compounds obtained withRH24K and RH24 at different degrees of biotin functionalization. FIG. 4relates to the results obtained with a monoclonal antibody, the testbeing performed on a microtitration plate. FIG. 5 represents the resultsobtained on serum with the Vidas immunoanalysis automated machine fromBioMérieux.

[0072] RH24K gives a bigger signal than RH24, irrespective of theexperimental conditions tested. An optimum biotin/p24 ratio of 25/1 forthe monoclonal antibodies, and of 10/1 for the serum, is observed.

EXAMPLE 6 Influence of the Composition, in Terms of Lysine Residues ofthe Polylysine Tag, on the Coupling Efficacy

[0073] The biological material used to evaluate the influence of thecomposition as lysine residues of the polylysine tag on the couplingefficacy is as follows:

[0074] the recombinant protein RH24K; this contains, in itsamino-terminal part, a polyhistidine tag [MRGS(H)₆GSVDESM] which servesto purify it by chelation with a metal ion, and, in its carboxy-terminalpart, a polylysine tag [PG(K)₆SVDESL] dedicated to the coupling; thisprotein can be represented as follows:

MRGS(H)₆GSVDESM-p24-PG(K)₆SVDESL)

[0075] the recombinant protein RHK24; this contains, in itsamino-terminal part, a polylysine tag [MRGSCH(K)₂HH(K)₂HH(K)₂GSVDESM];this protein can be represented as follows:

MRGSCH(K)₂HH(K)₂HH(K)₂GSVDESM-p24

[0076] the recombinant protein R24; this contains neither thecarboxy-terminal polylysine tag nor the amino-terminal polyhistidinetag; it can be represented by p24.

[0077] The polymers used are MAVE (maleic anhydride -co-methyl vinylether). The coupling conditions are identical for the three proteins.

[0078] Two coupling buffers were used, the 0.1 M, pH 8.2 carbonatebuffer and the 0.05 M, pH 9.0 Tris HCl buffer.

[0079] The results are given in the following table illustrating theyield for coupling the recombinant proteins with the MAVE polymercarried out under two different reaction conditions. They show theimportance of the contiguity of the lysine residues.

[0080] The reason for this is that the coupling yield obtained with theprotein RH24K (6 contiguous lysine residues) is very much greater thanthe coupling yield obtained with protein RHK24 (6 lysine residues inthree blocks of 2).

[0081] In point of fact, the coupling yield obtained with the proteinRHK24 (6 lysine residues in three blocks of 2) is similar to thatobtained with the protein R24 (no tag). TABLE Protein R24 RH24K RHK24polylysine no PG (K) MRGSCH (K) ₂HH (K) tag tag ₆SVDESL ₂HH (K) ₂GSVDESM0.1 M pH 8.2 30% 95% 33% carbonate buffer 0.05 M pH 9.0 35% 100% 20%Tris HCl buffer

EXAMPLE 7 Production of Anti-polylysine Tag (K)₆SVDESL Antibodies

[0082] The biological material used to obtain the anti-tag antibodiescapable of recognizing this tag in the context of a fusion with arecombinant protein is as follows:

[0083] the recombinant protein RH24K; this contains, in itsamino-terminal part, a polyhistidine tag [MRGS(H)₆GSVDESM] which servesto purify it by chelation with a metal ion, and, in its carboxy-terminalpart, a polylysine tag [PG(K)₆SVDESL] which is dedicated to the couplingand represented by

MRGS(H)₆GSVDESM-p24-PG(K)₆SVDESL

[0084] the peptide P400, the sequence of which is C(K)₆SVDESL, coupledto KLH (P400-KLH).

[0085] The biological material used to select the antibodies produced isas follows:

[0086] the recombinant protein RH24K

[0087] the recombinant protein RH24 containing no carboxy-terminalpolylysine tag, and represented by

MRGS(H)₆GSVDESM-p24)

[0088] the recombinant protein RH24; this contains neither thecarboxy-terminal polylysine tag nor the amino-terminal polyhistidinetag, and is represented by p24.

[0089] the peptide P400, the sequence of which is C(K)₆SVDESL.

[0090] The following two test formats were used to select the monoclonalantibodies:

[0091] a sandwich ELISA test, comprising, in the capture phase, ananti-mouse goat antibody, and, in the detection phase, the antigensRH24K or R24 revealed by an anti-24 monoclonal antibody coupled toperoxidase;

[0092] an indirect ELISA test, comprising, in the capture phase, theantigens RH24K or R24 or P400, and, in the detection phase, ananti-mouse IgV goat antibody coupled to peroxidase.

[0093] BALB/c and A/J mice were immunized according to the followingprotocol: 3 intra-peritoneal injections with, for the first injection,the protein RH24K, and, for the following two injections, the peptide400 coupled to KLH. Fusions were carried out and screenings carried outusing the sandwich and indirect ELISA tests described above.

[0094] 80 hybrids were selected according to the following criteria:

[0095] recognition of the peptide P400 (C(K)₆SVDESL)

[0096] recognition of the protein RH24K(MRGS(H)₆GSVDESM-p24-PG(K)₆SVDESL)

[0097] non-recognition of the protein RH24 (MRGS(H)₆GSVDESM-p24)

[0098] non-recognition of the protein R24.

[0099] These antibodies thus recognize the sequence (K)₆SVDESL fused toa protein, but not the sequence MRGS(H)₆GSVDESM also containing theSVDESL unit, nor the protein p24.

[0100] Five of these hybrids were cloned and produced in the form ofascites: these are the antibodies IG2D4, 2G2B3, 2G4A12, 5F12ES and14E1G7, all of IgG1 k isotype.

[0101] The results of the sandwich and indirect ELISA tests on the IG2D4antibodies are illustrated in FIGS. 6 and 7. FIG. 6 illustrates asandwich ELISA test and represents the OD as a function of the titer(dilution) of the IG2D4 antibody ascite; the recombinant proteins RH24Kand R24, constituting the detection phase, are used at a concentrationof 0.1 μg/ml. FIG. 7 illustrates an indirect ELISA test and representsthe OD as a function of the titer (dilution)(of the IG2D4 antibodyascite; the recombinant proteins RH24K and R24, constituting the capturephase, are used at a concentration of 0.5 μg/ml and the syntheticpeptide P400 is used at a concentration of 0.05 μg/ml.

Bibliography

[0102] (1) Porath J., Carlsson., Olsson., Belfrage J., Nature, 258, 598(1975)

[0103] (2) Porath J., Trends Anal. Chem., 7, 254 (1988)

[0104] (3) Cheynet, Protein Expression and Purification, 4, 367-372(1993)

1. Method for isolating a target biological material contained in asample, according to which a capture phase is provided, said targetbiological material is placed in contact with at least the capturephase, and the target biological material bound to the capture phase isdetected, said method being characterized in that the capture phasecomprises an organic molecule containing at least one reactive functionand at least one protein material capable of recognizing or binding,specifically and directly or indirectly, to the target biologicalmaterial, said protein material containing a specific site for covalentbinding to the reactive function of the organic molecule, which consistsof at least one tag comprising at least six contiguous lysine orlysine-based residues.
 2. Method according to claim 1, characterized inthat the capture phase also comprises a label.
 3. Method according toclaim 2, characterized in that the capture phase is a detection phase.4. Method according to claim 1, characterized in that a detection phaseis also provided, which comprises an organic molecule containing atleast one reactive function, at least one protein material capable ofrecognizing or binding, specifically and directly or indirectly, to thetarget biological material, and a label, said protein materialcontaining a specific site for covalent binding to the reactive functionof the organic molecule, which consists of at least one tag comprisingat least six contiguous lysine or lysine-based residues.
 5. Methodaccording to claim 4, characterized in that the organic molecule and theprotein material in the detection phase are, respectively, identical toand/or different from the organic molecule and the protein material inthe capture phase.
 6. Method according to claims 1 and/or 4,characterized in that the organic molecule is a particulate or linearpolymer.
 7. Method according to claim 6, characterized in that thepolymer is chosen from homopolymers such as polylysine or polytyrosine;and copolymers such as maleic anhydride copolymers, N-vinylpyrrolidonecopolymers, natural or synthetic polysaccharides and polynucleotides andamino acid copolymers such as enzymes.
 8. Method according to claim 7,characterized in that the polymer is chosen from maleic anhydride/methylvinyl ether copolymer, N-vinylpyrrolidone/N-acryloxysuccinimidecopolymer and poly-6-aminoglucose.
 9. Method according to claims 1and/or 4, characterized in that the organic molecule is a hapten. 10.Method according to claim 9, characterized in that the organic moleculeis bound to a carrier molecule.
 11. Method according to claim 9,characterized in that the organic molecule is biotin and the carriermolecule is avidin.
 12. Method according to claim 4, characterized inthat the organic molecule in the detection phase is the label. 13.Method according to claims 1 and/or 4, characterized in that the tag isplaced at the N- or C-terminal end of the protein material.
 14. Methodaccording to claims 1 and/or 4, characterized in that the reactivefunction of the organic molecule is chosen from ester, acid,halocarbonyl, sulfhydryl, disulfide, epoxide, haloalkyl and aldehydefunctions.
 15. Method according to any one of claims 1 to 14,characterized in that the protein material comprises an antigen and thebiological material is an antibody which is specifically recognized bysaid antigen.
 16. Method according to any one of claims 1 to 14,characterized in that the protein material comprises an antibody and thebiological material is an antigen which is specifically recognized bysaid antibody.
 17. Capture phase for a target biological material,characterized in that it comprises an organic molecule containing atleast one reactive function and at least one protein material capable ofrecognizing or of binding, specifically and directly or indirectly, tothe target biological material, said protein material containing aspecific site for covalent binding to the reactive function of theorganic molecule, which consists of at least one tag comprising at leastsix contiguous lysine or lysine-based residues.
 18. Detection phase fora target biological material, characterized in that it comprises anorganic molecule containing at least one reactive function, at least oneprotein material capable of recognizing or of binding, specifically anddirectly or indirectly, to the target biological material, and a label,said protein material containing a specific site for covalent binding tothe reactive function of the organic molecule, which consists of atleast one tag comprising at least six contiguous lysine or lysine-basedresidues.
 19. Capture or detection phase according to claim 17 or 18,characterized in that the organic molecule is a particulate or linearpolymer.
 20. Capture or detection phase according to claim 19,characterized in that the polymer is chosen from homopolymers such aspolylysine or polytyrosine; and copolymers such as maleic anhydridecopolymers, N-vinylpyrrolidone copolymers, natural or syntheticpolysaccharides and polynucleotides and amino acid copolymers such asenzymes.
 21. Capture or detection phase according to claim 20,characterized in that the polymer is chosen from maleic anhydride/methylvinyl ether copolymer, N-vinylpyrrolidone/N-acryloxysuccinimidecopolymer, poly-6-aminoglucose, horseradish peroxidase (HRP) andalkaline phosphatase.
 22. Capture phase according to claim 21,characterized in that the organic molecule is a hapten, such as biotin.23. Capture or detection phase according to claim 17 or 18,characterized in that the tag is placed at the N- or C-terminal end ofthe protein material.
 24. Capture or detection phase according to claim17 or 18, characterized in that the reactive function of the organicmolecule is chosen from ester, acid, halocarbonyl, sulfhydryl,disulfide, epoxide, haloalkyl and aldehyde functions.
 25. Detectionphase according to claim 18, characterized in that the organic moleculeis the label.
 26. Detection phase according to claim 18 or 25,characterized in that the label is chosen from the group consisting ofan enzyme, a protein, a peptide, an antibody, a hapten such as biotin oriminobiotin, a fluorescent compound such as rhodamine, a radioactivecompound, a chemiluminescent compound, an electron-density component, amagnetic component and analogs.
 27. Reagent for isolating a targetbiological material, comprising a capture phase according to claim 17and any one of claims 19 to 24, and/or a detection phase according toany one of claims 18 to
 26. 28. Reagent according to claim 27,characterized in that the capture phase is bound, directly orindirectly, to a solid support, by passive adsorption or by covalency.